This application is the U.S. national phase of PCT/AT98/00046, filed Feb. 27, 1998, which claims priority to Austrian Application A336/97, filed Feb. 27, 1997.
The invention relates to factor Xxcex94 analogues having a deletion of the amino acids from Arg180 to Arg234 and a modification in the region of the amino acid sequence between Gly173 and Arg179, to preparations containing the factor Xxcex94 analogues or factor Xa analogues according to the invention, as well as to methods of preparing the factor Xxcex94 analogues according to the invention.
After the blood coagulation process has been initiated, the coagulation cascade continues through sequential activation of various proenzymes (zymogens) in the blood to their active forms, the serine proteases. Among them are, inter alia, factor XII/XIIa, factor XI/XIa, factor IX/IXa, factor.,X/Xa, factor VII/VIIa and prothrombin/thrombin. In their physiological state, most of these enzymes are only active if associated to a membrane surface in a complex. Ca ions are involved in many of these processes. The blood coagulation will either follow the intrinsic pathway, wherein all protein components are present in the blood, or the extrinsic pathway, wherein the tissue factor plays a critical role. Finally, the wound will close by thrombin cleaving fibrinogen to fibrin.
The prothrombinase complex is responsible for activating prothrombin to thrombin. Thrombin is an important enzyme which can act as a procoagulant as well as an anticoagulant. The prothrombinase complex, in which, inter alia, factor Va (as cofactor) and factor Xa (as serine protease) are involved, assembles in a Ca-dependent association at the surface of phospholipids. It is discussed that factor Xa is the catalytic component of the prothrombinase complex.
Factor X (Stuart-Prower factor) is a vitamin K-dependent coagulation glycoprotein which can be activated by the intrinsic and the extrinsic blood coagulation cascade. The primary translation product of factor X (pre-pro-FX) has 488 amino acids and is synthesized by the liver or human hepatoma cells initially as a single chain 75 kD precursor protein. In plasma, factor X is largely present as a double chain molecule (Fair et al., 1984, Blood 64:194-204).
During biosynthesis, after cleavage of the pre-sequence by a signal peptidase (between Ser23/Leu24) and of the propeptide (between Arg40/Ala41), the single chain factor X molecule is cleaved by processing and removal of the tripeptide Arg180-Lys181-Arg182 to the double chain form consisting of the approximately 22 kD light chain and the approximately 50 kD heavy chain, which are connected via a disulfide bridge (FIG. 2A, Panel 1A). Therefore, factor X circulates in the plasma as a double chain molecule.
During the blood coagulation process, factor X is converted from inactive zymogen to active protease factor Xa by limited proteolysis, wherein factor X can be activated to factor Xa in either of two membrane-associated complexes: in the extrinsic factor VIIa-tissue factor complex or in the intrinsic factor VIIIa-factor IXa-phospholipid-Ca complex, or xe2x80x9ctenase complexxe2x80x9d (Mertens et al., 1980, Biochem. J. 185:647-658). A proteolytic cleavage between amino acids Arg234/Ile235 results in the release of an activation peptide having a length of 52 amino acids from the N-terminus of the heavy chain and thus to the formation of the active enzyme, factor Xa. The catalytic center of factor Xa is located on the heavy chain.
Activation via the factor VIIa-TF (extrinsic) complex results in the formation of Factor Xaxcex1 (35 kD) and factor Xaxcex2 (31 kD), with a polypeptide of 42 (kD) forming, too, if the factor VIIa,concentration in the complex is low. Factor Xaxcex1 is formed by a cleavage at Arg234/Ile235 of the heavy chain and represents the activation of factor X to factor Xa. The occurence of factor Xaxcex2 presumably results from an autocatalytic cleavage at Arg469/Gly470 in the C-terminus of the heavy chain of factor Xaxcex1 and the cleavage of a 4.5 kD peptide. Like factor Xaxcex1, factor Xaxcex2 has catalytic activity. It has been shown, however, that a plasminogen receptor binding site is formed by the cleavage of factor Xaxcex1 to factor Xaxcex2, and that factor Xaxcex2 optionally has fibrinolytic activity or is involved in fibrinolysis as a cofactor. The transformation of factor Xaxcex1 to factor Xaxcex2, however, is slower than the formation of thrombin, thus preventing the initiation of fibrinolysis before a blood clot is formed (Pryzdial et al., 1996, J. Biol. Chem. 271:16614-16620; Pryzdial et al., 1996, J. Biol. Chem. 271:16621-16626).
The 42 kD polypeptide results from processing in the C-terminus of the heavy chain between Arg469/Gly470 without previous processing between Arg234/Ile235. Like a factor Xaxcex3 fragment formed by proteolysis at Lys370, this intermediate has no catalytic activity (Mertens et al., 1980, Biochem. J. 185:647-658; Pryzdial et al., 1996, J. Biol. Chem. 271:16614-16620).
Intrinsic factor X activation is catalysed by the factor IXa-factor VIIIa complex. The same processing products are obtained during activation, but the factor Xaxcex2 product is obtained in a larger quantity than other factor X processing products (Jesty et al., 1974, J. Biol. Chem. 249:5614).
In vitro, factor X can, for instance, be activated by Russell""s viper venom (RVV) or trypsin (Bajaj et al., 1973, J. Biol. Chem. 248:7729-7741) or by purified physiological activators, such as FVIIa/TF complex or factor IXa/factor VIIIa complex (Mertens et al., 1980, Biochem. J. 185:647-658).
Most commercially available factor X products from plasma contain a mixture of factor Xaxcex1 and factor Xaxcex2, because after activation of factor X to factor Xa mainly factor Xaxcex1 is formed, which is, in turn, cleaved to factor Xaxcex2 in an autocatalytic process.
In order to produce a uniform factor Xa product having high molecular integrity, EP 0 651 054 suggested to activate factor X with RVV over an extended period of time so that the resulting final product substantially contains factor Xaxcex2. The by-products, e.g. factor Xaxcex1, as well as the protease were subsequently removed by several chromatographic steps.
cDNA for factor X has been isolated and characterized (Leytus et al., 1984, Proc. Natl. Acad. Sci., U.S.A., 82:3699-3702; Fung et al., 1985, Proc. Natl. Acad. Sci., U.S.A., 82:3591-3595). Human factor X has been expressed in vitro in various types of cells, such as human embryonal renal cells or CHO cells (Rudolph et al., 1997, Prot. Expr. Purif. 10:373-378, Wolf et al., 1991, J. Biol. Chem. 266:13726-13730). However, it was found that in the recombinant expression of human factor X, the processing at position Arg40/Ala41 is inefficient, as opposed to the situation in viva, and that different N-termini form at the light chain of factor X (Wolf et al., 1991, J. Biol. Chem. 266:13726-13730). Recombinant factor X (rFX) was activated to rfactor Xa (rFXa) by RVV in vitro, or rFXa was expressed directly, with the activation peptide being deleted from amino acid 183 to amino acid 234 and replaced by a tripeptide in order to allow processing directly to a double chain rFXa form. About 70% of purified rFX was processed to light and heavy chain, while the remaining 300 represented single chain rFX of 75 kD. Direct expression of rFXa did result in the formation of active factor Xa, but also of inactive intermediates. Furthermore, Wolf et al. (1991, J. Biol. Chem. 266:13726-13730) detected still reduced activity of recombinant factor X, which they ascribed to the poorer ability of rFX to be activated by RVV and to the inactive protein and polypeptide populations of the single chain precursor molecule. In particular, they found high rFXa instability when expressed by recombinant cells, which they ascribed to the high rate of autoproteolysis.
In order to study the function of the C-terminal peptide of factor Xaxcex1, Eby et al. (1992, Blood 80 (suppl. 1): 1214 A) introduced a stop codon at position Gly430 of the factor X sequence. However, they did not find a difference between the rate of activation of factor Xa (Fxaxcex1) with xcex2-peptide or a deletion mutant without xcex2-peptide (FXaxcex2).
Factor Xa is an important component of the prothrombinase complex and is therefore under discussion as a primary mediator for quick hemostasis, and thus it seems suitable for the treatment of patients suffering from blood coagulation disorders, e.g. hemophilia.
Particularly the treatment of hemophilia patients suffering from factor VIII or factor IX deficiency with factor concentrates produced from plasma is often complicated by the formation of inhibiting antibodies against these factors in long-term therapy. Therefore, a number of alternatives have been developed to treat hemophiliacs with factors having bypass activity. The use of prothrombin complex concentrate, partially activated prothrombinase complex (APPC), factor VIIa or FEIBA has been suggested. Commercial preparations having factor VIII bypass activity (FEIBA) are, for instance, FEIBA(copyright) or Autoplex(copyright). FEIBA, contains comparable units of factor II, factor VII, factor IX, factor X and FEIBA, small amounts of factor VIII and factor V, and traces of activated coagulation factors, such as thrombin and factor Xa or a factor having factor X-like activity (Elsinger, 1982, Activated Prothrombin Complex Concentrates. Ed. Mariani, Russo, Mandelli, pp. 77-87). Elsinger particularly points at the importance of a xe2x80x9cfactor Xa-likexe2x80x9d activity in FEIBA. Factor VIII bypass activity was shown by Giles et al (1988, British J. Haematology 9:491-497) for a combination of purified factor Xa and phospholipids in an animal model.
Therefore, factor X/Xa or factor X/Xa-like proteins, either alone or as a component of a coagulation complex, are in high demand and can be used in various fields of application in hemostasis therapy.
In vivo as well as in vitro, the half-life of factor Xa is considerably shorter than the half-life of the zymogen. For instance, factor X can be stored stably in glycerol for 18 months, while factor Xa is stable for only 5 months under the same conditions (Bajaj et al., 1973, J. Biol. Chem. 248:7729-7741) and shows reduced activity by more than 60% after 8 months in glycerol at 4xc2x0 C. (Teng et al., 1981, Thrombosis Res. 22:213-220). The half-life of factor Xa in serum is a mere 30 seconds.
Because factor X is instable, the administration of factor X preparations has been suggested (U.S. Pat. No. 4,501,731). If, however, the bleeding is so serious that the patient might die, particularly in a hemophiliac, the administration of factor X is ineffective, because owing to the functional xe2x80x9ctenase complexxe2x80x9d deficiency in the intrinsic pathway of blood coagulation, factor X can not be sufficiently activated to factor Xa, and activation via the extrinsic pathway is often too slow to show effects quickly. Moreover, hemophiliacs have sufficient amounts of factor X, but its prothrombinase activity is 1000 times less than that of factor Xa. In such cases it is necessary to administer activated factor Xa directly, optionally in combination with phospholipids, as described in Giles et al. (1988, British J. Haematology 9:491-497) or with other coagulation factors, e.g. with factor VIII bypass activity.
In the preparation of factor Xa from factor X, activation so far mostly has been carried out by non-physiological activators of animal origin, such as RVV or trypsin, and it was necessary to make absolutely sure that the final product is completely free of these proteases. As mentioned above, when factor X is activated to factor Xa, quite a number of intermediates, some of them inactive, are formed (Bajaj et al., 1973, J. Bio. Chem. 248:7729-7741; Mertens et al., 1980, Biochem. J. 185:647-658). The presence of such intermediates results in reduced specific activity of the product and may produce intermediates which can function as active serine protease antagonists. Therefore, the.preparation of a uniform, pure product having high specific activity according to conventional methods requires complex processes of activation and chromatographic purification.
Thus, the aim of the present invention is to provide a preparation containing a polypeptide having factor X/Xa activity which exhibits high stability and can be activated to factor Xa without using any of the usual proteases, particularly those of animal origin, such as, for instance, RVV or trypsin. Another aim is to provide a pharmaceutical preparation having factor VIII bypass activity.
According to the present invention, the aim is reached by providing a factor X analogue having a deletion of the amino acids Arg180 to Arg234 of the factor X amino acid sequence and a modification of this factor X deletion mutant in the region of the amino acid sequence between Gly173 and Arg179. By the deletion of the amino acid sequence from Arg180 to Arg234, the tripeptide Arg180 to Arg182 as well as the activation peptide Ser183 to Arg234 are deleted, and the light and heavy chains of factor X and the amino acids Arg179 and Ile235 are directly fused. This fusion sequence, however, does not contain a natural cleavage site for a protease. By modifying the region of the factor X sequence between amino acid Gly173 and Arg179 and optionally of Ile235, a factor X deletion mutant according to the present invention is obtained, which has a novel detection and processing site not occurring at this position in the polypeptide for a protease which would not usually cleave the polypeptide at this position. Said modification is, at least, an exchange of at least one amino acid between position Gly173 and Arg179 and, optionally of Ile235 of the factor X amino acid sequence. The position of amino acids refers to the numbering according to the sequence presented in FIGS. 1A and 1B, starting with Met1 and ending with Lys488. In order to simplify the nomenclature, the amino acid numbering given for the complete factor X sequence is adhered to for the modified factor X deletion mutant according to the present invention, but said modified factor X deletion mutant will hereinafter be referred to as factor Xxcex94 analogue.
Said modification can be a substitution of at least one amino acid, or an insertion of a peptide sequence representing a protease recognition or cleavage site. In the factor Xxcex94 analogue according to the present invention, the modification is preferably such that it represents a recognition and cleavage sequence for a protease from the group of endoproteases, such as kexin/Kex2, furin/PACE, PC1/PC3, PC2, PC4, PACE 4, LPC/PC7 (as described in Barr et al., 1991, Cell 66:1-3 or in U.S. Pat. No. 5,460,950), serine proteases, such as factor IIa, factor VIIa, factor IXa, factor XIIa, factor XIa, factor Xa, or kallikrein, or a derivative of these proteases.
Preferably, said modification is selected such that processing by one of these proteases leads to a polypeptide corresponding to native factor Xa in its biological activity and displaying factor Xa activity. For optimal processing, it may be necessary in individual cases to exchange the amino acid Ile235, too. Preferably, however, the NH2-terminal amino acid isoleucine of the heavy chain should still be maintained after activation, because isoleucine represents one of those amino acids which perform an essential function in the formation of the substrate binding pocket (Watzke. et al., 1995, Molecular Basis of Thrombosis and Hemostasis, ed. Katherine High and Harold Roberts). The factor Xxcex94 analogues according to the present invention display a structural difference, particularly on the amino acid level, as compared to a native factor X sequence, but after activation their activity is comparable to that of naturally occurring factor X or factor Xa, respectively.